The overarching goal of this application is to create tools and efficient methods to define genes that can promote human health. While a tremendous amount of data has been cataloged on gene mutation and changes in gene expression associated with complex human disease, our understanding of those genes that could be co-opted to restore patient health is lacking. To address this need and test for genes that when restored to wild type function promote health, we propose develop mutagenic, revertible and conditional alleles that provide spatial and temporal control of gene expression. The ability to make site-specific, untagged mutant alleles in zebrafish and other models has been greatly advanced by custom nucleases that include TALENs and CRISPR/Cas9 systems. These systems operate on the same principle: they are designed to bind to specific sequences in the genome and create a double strand break. The goals of this proposal leverage the activities of TALEN and CRISPR/Cas9 technologies to make site-specific double strand breaks. These tools and techniques will have direct implications for providing precise gene editing techniques to assess the roles of genes in disease and their ability to promote health following disease progression. While we will develop these methodologies in zebrafish due to their ease of gene delivery, we anticipate these methodologies will not only enhance the efficiency of gene editing but will be readily adaptable for use in other model organisms and large animals. In our opinion, this will have important implications for modeling human disease and health in animal systems by greatly enhancing the ability to make predictable alleles, small nucleotide polymorphisms similar to those associated with human disease, and conditional alleles to test for the ability of a gene to restore health.